Plasmids are extrachromosomal genes of cyclic DNA found in microorganism cells. Plasmids are currently being used as a means of genetic recombination of microorganisms and are becoming increasingly important in research in the fermentation industry.
Studies have recently been made in the plasmids containing foreign DNA coding for metabolic products or for particular growth requisites of microorganisms, such as amino acids or peptides. Some plasmids have been introduced into host microorganisms to obtain transformants.
The present inventors succeeded in constructing a novel plasmid into which DNA coding for the secretion (extracellular production) of penicillinase was inserted. This DNA was prepared from chromosomal DNA of a microorganism belonging to the genus Bacillus. The present inventors further succeeded in obtaining a novel and useful transformant, Escherichia coli HB101 (pEAP2), by introducing the above plasmid into the HB101 strain of Escherichia coli (Japanese Patent Publication (unexamined) 162886/1984).
Further, the present inventors succeeded in constructing a novel plasmid into which DNA coding for the secretion of xylanase was inserted, which DNA was prepared from chromosomal DNA of a microorganism belonging to the genus Bacillus. A novel and useful transformant, Escherichia coli HB101 (pCX 311) was then obtained by introducing the above plasmid into the HB101 strain of Escherichia coli (Japanese Patent Publication (unexamined) 126085/1985).
Then, the present inventors succeeded in constructing a novel plasmid by inserting a DNA fragment coding for production of a desired useful, physiologically active substance into a plasmid in which DNA coding for secretion of penicillinase had been inserted and, further, succeeded in obtaining a novel and useful transformant, Escherichia coli HB101 (pXP102-3), by introducing the above plasmid into the HB101 strain of Escherichia coli. (Japanese Patent Application 278681/1984).
Methods as mentioned above are very useful where a plasmid having an inserted DNA fragment coding for the secretion of a useful, physiologically active substance is introduced into a host organism to transform the host organism. The resultant transformant is cultured to allow for secretion and accumulation of the useful, physiologically active substance outside the cell, which substance is then isolated and collected. Advantages of such methods include maintenance of transformant viability, the absence of intracellular substance accumulation with feed-back inhibition of substance production and easy purification of the secreted substance.
At present, only a limited number of microorganisms are known to secrete desirable, useful, physiologically active substances outside a cell.
Escherichia coli (E. coli), which has been widely used as a host organism in gene recombination, and which has been studied in detail, is not known to primarily secrete its product outside the cells. The same properties are exhibited by transformants of E. coli.
On the other hand, Bacillus subtilis, which is one of the typical bacterial of the genus Bacillus, produces and secretes enzymes. However, it is genetically unstable, i.e., its genotype has a tendency to change. Further, its products are often decomposed by a protease produced by the microorganism itself.
Accordingly, microorganisms capable of producing and secreting desired, useful, physiologically active substances have great utility in industry. In particular, there is a need for a new transformant of E. coli, which is capable of secreting a product.
The present inventors have investigated the question of what gene plays a role in controlling extracellular secretion and have identified both a DNA region capable of inducing the extracellular secretion of useful, physiologically active substances in a transformed host and a promoter DNA region, which regulates expression of the former DNA region. Further, the inventors have succeeded in constructing a novel plasmid having these DNA regions and obtaining a novel microorganism transformed with the novel plasmid.